Treatment of polycystic diseases with an hdac6 inhibitor

ABSTRACT

An HDAC6-specific inhibitor (i.e., a compound of Formula I or II) is shown to reduce the pathogenesis associated with polycystic disease. Administration of an HDAC6-specific inhibitor attenuated many of the symptoms characteristic of polycystic liver disease including cyst formation, cyst growth and cholangiocyte proliferation. Treatment with a HDAC6-specific inhibitor also increased the amount of bile duct acetylated tubulin and β-catenin phosphorylation and/or acetylation while reducing bile duct β-catenin synthesis. These results demonstrate that HDAC6 is overexpressed in cystic cholangiocytes and that its pharmacological inhibition reduces cholangiocyte proliferation and cyst growth.

RELATED APPLICATIONS

This application claims priority to and the benefit of U.S. ProvisionalApplication No. 61/895,223, filed Oct. 24, 2013, which is herebyincorporated by reference in its entirety.

GOVERNMENT FUNDING

This invention was made with government support under P30DK084567,R21CA166635 and R01 DK24031 awarded by the National Institutes of Health(NIH). The government has certain rights in the invention.

TECHNICAL FIELD

This disclosure reports on the administration of HDAC6 inhibitors forthe treatment of polycystic diseases.

BACKGROUND

Post-translational modification of proteins through acetylation anddeacetylation of lysine residues plays a critical role in regulating avariety of cellular functions, including the control of cell shape,differentiation and proliferation. Histone deacetylases (HDACs) arezinc-binding hydrolases that catalyze the deacetylation of lysineresidues on histones as well as non-histone proteins (Haberland et alNature Rev. Genet. 2009, 10, 32-42). Eleven Zn binding human HDACs havebeen identified (Taunton et al. Science 1996, 272, 408-411; Yang et al.J. Biol. Chem. 1997, 272, 28001-28007; Grozinger et al. Proc. Natl.Acad. Sd. U.S.A. 1999, 96, 4868-4873; Kao et al. Genes Dev. 2000, 14,55-66. Hu et al J. Biol. Chem. 2000, 275, 15254-15264; Zhou et al. Proc.Natl. Acad. Sci U.S.A. 2001, 98, 10572-10577; Venter et al. Science2001, 291, 1304-1351). These members are classified into four families:Class I (HDAC1, 2 and 3), Class IIa (HDAC4, 5, 7 and 9), Class IIb(HDAC6 and 10) and Class IV (HDAC11).

Class I HDACs (HDACs 1, 2 and 3) modulate gene expression throughdeacetylation of the N-acetyl-lysine residues of histone proteins andother transcriptional regulators in the nucleus of the cell (Hassig etal. Curr. Opin. Chem. Biol. 1997, 1, 300-308).

HDAC6, a class IIb HDAC, is unique amongst the zinc dependent HDACs inhumans. Located in the cytoplasm, HDAC6 has two catalytic domains and anubiquitin binding domain in its C terminal region. The substrates ofHDAC 6 include tubulin, peroxiredoxin, cortactin and heat shock protein90 (hsp90) but not histones. HDAC6 plays a key role in microtubuledynamics including cell migration and cell-cell interactions and it isrequired for aggresome formation with ubiquitinated proteins.

Provided herein are small molecule inhibitors of HDAC6, pharmaceuticalcompositions thereof, and methods of using these compounds to treatpolycystic diseases.

SUMMARY OF THE INVENTION

This disclosure provides for methods of treating polycystic diseasecomprising administering to a subject with polycystic disease atherapeutically effective amount of a histone deacetylase 6 (HDAC6)specific inhibitor compound of Formula I:

or a pharmaceutically acceptable salt thereof,

wherein ring B is aryl or heteroaryl, R₁ is an aryl or heteroaryl, eachof which may be optionally substituted by OH, halo, or C₁₋₆-alkyl, and Ris H or C₁₋₆-alkyl, and wherein the amount of the histone deacetylase 6(HDAC6) specific inhibitor compound of Formula I is effective atreducing cyst growth in the subject.

This disclosure also provides for methods of treating polycystic diseasecomprising administering to a subject with polycystic disease atherapeutically effective amount of a histone deacetylase 6 (HDAC6)specific inhibitor compound of Formula II:

or a pharmaceutically acceptable salt thereof,

wherein,

X is C or O;

R_(y) is independently, at each occurrence, selected from the groupconsisting of C₁₋₆-alkyl, C₁₋₆-alkoxy, halo, —C₁₋₆ haloalkyl, —C₁₋₆dihaloalkyl, —C₁₋₆ trihaloalkyl, —OH, —N(R¹)₂, —C(O)R′, —CO₂R¹, and—C(O)N(R¹)₂;

or:

two R_(y) groups on the same or adjacent carbon atoms are taken togetherto form a C₃₋₈-cycloalkyl or C₃₋₇-heterocycloalkyl ring, each of whichmay be fused or isolated;

R_(z) is independently, at each occurrence, selected from the groupconsisting of C₁₋₆-alkyl, C₁₋₆-alkoxy, halo, —C₁₋₆ haloalkyl, —C₁₋₆dihaloalkyl, —C₁₋₆ trihaloalkyl, —OH, —N(R²)₂, —C(O)R², —CO₂R²,—C(O)N(R²)₂,

each R¹ is independently, at each occurrence, selected from the groupconsisting of H, C₁₋₆-alkyl, C₃₋₈-cycloalkyl, C₃₋₇-heterocycloalkyl,aryl, heteroaryl, C₁₋₆-alkyl-cycloalkyl, C₁₋₆-alkyl-heterocycloalkyl,C₁₋₆-alkyl-aryl, and C₁₋₆-alkyl-heteroaryl;

each R² is independently, at each occurrence, selected from the groupconsisting of H or C₁₋₆-alkyl;

m is 0, 1, 2, or 3; and

n is 0, 1, 2, or 3.

and wherein the amount of the histone deacetylase 6 (HDAC6) specificinhibitor compound of Formula II is effective at reducing cyst growth inthe subject.

In certain embodiments, the histone deacetylase 6 (HDAC6) specificinhibitor compound prevents the formation of cysts in the subject.

In certain embodiments, the cysts can be located in the liver and/orkidney.

In certain embodiments, the subject has a mutation in at least one ofthe PRKCSH (Protein Kinase C Substrate 80K-H) and Sec63 genes.

In certain embodiments, the polycystic disease is renal cystic disease.

In certain embodiments, the polycystic disease is the polycystic kidneydisease.

In certain embodiments, the polycystic disease is an autosomal dominantpolycystic kidney disease (ADPKD). The subject can have a mutation in atleast one of the Pkd1 and Pkd2 genes.

In certain embodiments, the polycystic disease is an autosomal recessivepolycystic kidney disease (ARPKD). The subject can have a mutation inthe Pkhd1 gene.

In certain embodiments, the amount of the HDAC6 specific inhibitorcompound is effective at inhibiting cholangiocyte proliferation and/orbile duct β-catenin synthesis and/or promoting bile duct β-cateninphosphorylation and/or acetylation.

In certain embodiments, the amount of the HDAC6 specific inhibitorcompound is effective at increasing the amount of bile duct acetylatedtubulin and/or decreasing bile duct β-catenin synthesis.

In certain embodiments, the histone deacetylase 6 (HDAC6) specificinhibitor compound of Formula I is Compound A-1 having the structure:

or a pharmaceutically acceptable salt thereof,

or Compound B-1 having the structure:

or a pharmaceutically acceptable salt thereof.

In certain embodiments, the amount of the histone deacetylase 6 (HDAC6)specific inhibitor compounds A-1 and B-1 are effective at reducing cystgrowth in a subject.

In certain embodiments, the amount of the histone deacetylase 6 (HDAC6)specific inhibitor compounds A-1 and B-1 are effective at inhibitingcholangiocyte proliferation.

In certain embodiments, the histone deacetylase 6 (HDAC6) specificinhibitor compound of Formula II is Compound C-2 having the structure:

or a pharmaceutically acceptable salt thereof.

In certain embodiments, the amount of the histone deacetylase 6 (HDAC6)specific inhibitor compound C-2 is effective at reducing cyst growth ina subject.

In certain embodiments, the amount of the histone deacetylase 6 (HDAC6)specific inhibitor compound C-2 is effective at inhibiting cholangiocyteproliferation.

In certain embodiments, the disclosure teaches a kit comprising atherapeutically effective amount of HDAC6 specific inhibitor compoundsof Formula I or II and instructions for use in treating a polycysticdisease.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1C shows an analysis of HDAC6 expression in cysticcholangiocytes. FIG. 1A: PCR analysis showing HDAC6 mRNA expression inboth normal (NRC) and PCK rats cholangiocytes. FIG. 1B: RepresentativeWestern blot for HDAC6 and acetylated-α-tubulin expression in NRC andPCK rat cholangiocytes and densitometric analysis of the western blotresults. (* p<0.05, 6 n=3 for control, n=4 for PCK). FIG. 1C,Immunofluorescence confocal microscopy of liver cysts in PCK rats (n=5),human ADPKD (n=3) and ARPKD (n=3) livers and respective controls (n=5and 3 for rats and humans). HDAC6 is green, CK19 is red, and nuclei areblue (DAPI).

FIGS. 2A-2D shows HDAC6 inhibition decreases cystic cholangiocytesproliferation and cystic growth. Cells were incubated in 96-well platesand proliferation was analyzed by MTS assay. FIG. 2A shows the level ofPCK rat cholangiocytes proliferation over time in the presence ofdifferent doses of tubastatin-A. (*p<0.05). FIG. 2B shows tubastatin-Aand tubacin decrease the proliferation of PCK rat cholangiocytes over a3 day time period. (*p<0.05). FIG. 2C depicts representative images ofbiliary cysts freshly isolated from PCK rats. The cysts were embedded inrat tail collagen matrix and treated with two different HDAC6 specificinhibitors, tubastatin-A and tubacin. FIG. 2D shows a quantitativeanalysis of cyst growth over time and that both inhibitors (n=12 for 10uM tubastatinA, n=4 for 2 uM tubacin, n=16 for untreated controls.Magnification 40×. *p<0.05) significantly reduce cyst growth.

FIGS. 3A-3C shows HDAC6 inhibitors increase acetylated α-tubulin anddecrease β-catenin. PCK cholangiocytes were treated with 20 μMTubastatin A or 2 μM Tubacin, lysed and used for Western blotting. FIG.3A depicts representative images of western blots (n=3) demonstratingthat treatment with the HDAC6 inhibitors increases acetylated α-tubulinlevels while decreasing β-catenin, cyclin D1 and c-myc. The acetylationlevels of histone-3 remained unaffected. Actin was used as a loadingcontrol. FIG. 3B depicts representative images of Western blots (n=3)showing HDAC6 inhibitors increase the amount of phospho- andacetyl-β-catenin compared to untreated cells using total-β-catenin asloading control. FIG. 3C depicts Western blots and RT-PCR analysisshowing a decrease in β-catenin protein expression over time aftertreatment with 2 uM tubacin, while β-catenin mRNA remained stable. (*p<0.05).

FIGS. 4A-4C shows that the HDAC6 inhibitor, Compound A-1, inhibits cystgrowth in vivo. FIG. 4A depicts proliferation assays on normal (NRC) andcystic cholangiocytes (PCK) incubated in the presence of different dosesof Compound A-1. FIG. 4B depicts representative liver and kidneysections stained with picrosirius red from PCK rats treated with vehicle(n=8) or Compound A-1 (n=8). Bar=2500 μm. FIG. 4C, quantificationanalysis of cystic area expressed as percentage of total parenchymaarea. *p<0.05.

FIG. 5A depicts a human ADPKD 3-day proliferation assay showingdecreased proliferation induced by both HDAC6 specific inhibitors,tubastatin-A and tubacin.

FIG. 5B depicts a PCK cells proliferation analysis by cell countingshowing the effect of 20 μM tubastatin A.

FIG. 6A shows PCK cholangiocytes treated with the HDAC6-specificinhibitor compound A-1 and β-catenin expression analyzed byimmunofluorescence and compared to normal cells (NRC).

FIG. 6B depicts a β-catenin fluorescence quantification assessment thatshows an increased β-catenin expression in PCK cells both in nucleus andcytoplasm compared to NRC. The treatment with the HDAC6-specificinhibitor compound A-1 reduced β-catenin expression in PCK cells.

FIG. 7 shows the volume of cysts in the kidneys of male PCK-1 ratstreated with HDAC6 inhibitors of Formula I (compound B-1) and Formula II(Compound C-2)

DETAILED DESCRIPTION OF THE INVENTION Definitions

Listed below are definitions of various terms used to describe thisinvention. These definitions apply to the terms as they are usedthroughout this specification and claims, unless otherwise limited inspecific instances, either individually or as part of a larger group.

The term “about” generally indicates a possible variation of no morethan 10%, 5%, or 1% of a value. For example, “about 25 mg/kg” willgenerally indicate, in its broadest sense, a value of 22.5-27.5 mg/kg,i.e., 25±2.5 mg/kg.

The term “alkyl” refers to saturated, straight- or branched-chainhydrocarbon moieties containing, in certain embodiments, between one andsix, or one and eight carbon atoms, respectively. Examples of C₁-C₆alkyl moieties include, but are not limited to, methyl, ethyl, propyl,isopropyl, n-butyl, tert-butyl, neopentyl, n-hexyl moieties; andexamples of C₁-C₈ alkyl moieties include, but are not limited to,methyl, ethyl, propyl, isopropyl, n-butyl, tert-butyl, neopentyl,n-hexyl, heptyl, and octyl moieties.

The term “aryl” refers to a mono- or poly-cyclic carbocyclic ring systemhaving one or more aromatic rings, fused or non-fused, including, butnot limited to, phenyl, naphthyl, tetrahydronaphthyl, indanyl, idenyland the like. In some embodiments, aryl groups have 6 carbon atoms. Insome embodiments, aryl groups have from six to ten carbon atoms. In someembodiments, aryl groups have from six to sixteen carbon atoms.

The term “heteroaryl” refers to a mono- or poly-cyclic (e.g., bi-, ortri-cyclic or more) fused or non-fused moiety or ring system having atleast one aromatic ring, where one or more of the ring-forming atoms isa heteroatom such as oxygen, sulfur, or nitrogen. In some embodiments,the heteroaryl group has from about one to six carbon atoms, and infurther embodiments from one to fifteen carbon atoms. In someembodiments, the heteroaryl group contains five to sixteen ring atoms ofwhich one ring atom is selected from oxygen, sulfur, and nitrogen; zero,one, two, or three ring atoms are additional heteroatoms independentlyselected from oxygen, sulfur, and nitrogen; and the remaining ring atomsare carbon. Heteroaryl includes, but is not limited to, pyridinyl,pyrazinyl, pyrimidinyl, pyrrolyl, pyrazolyl, imidazolyl, thiazolyl,oxazolyl, isooxazolyl, thiazolyl, thiadiazolyl, oxadiazolyl, thiophenyl,furanyl, indolyl, quinolinyl, isoquinolinyl, benzimidazolyl,benzooxazolyl, quinoxalinyl, acridinyl, and the like.

The term “halo” refers to a halogen, such as fluorine, chlorine,bromine, and iodine.

The term “subject” as used herein refers to a mammal. A subjecttherefore refers to, for example, dogs, cats, horses, cows, pigs, guineapigs, and the like. Preferably the subject is a human. When the subjectis a human, the subject may be referred to herein as a patient.

As used herein, the terms “treating,” “treatment” and the like are usedherein to mean obtaining a desired pharmacologic and/or physiologiceffect that at least alleviates or abates a polycystic disease. Theeffect may be prophylactic in terms of completely or partiallypreventing a polycystic disease or sign or symptom thereof, and/or maybe therapeutic in terms of a partial or complete cure for a polycysticdisease and/or adverse effect attributable to the polycystic disease.“Treating” also covers any treatment of a polycystic disease in amammal, and includes: (a) preventing a polycystic disease from occurringin a subject that may be predisposed to a polycystic disease, but hasnot yet been diagnosed as having it; (b) inhibiting a polycysticdisease, i.e., arresting its development; or (c) relieving orameliorating the polycystic disease, e.g., cause regression of thepolycystic disease. As used herein, to “treat” includes systemicamelioration of the symptoms associated with the pathology and/or adelay in onset of symptoms. Clinical and sub-clinical evidence of“treatment” will vary with the pathology, the individual and thetreatment.

In certain embodiments, the terms “treating” or “treatment” can refer toreducing cholangiocyte proliferation, bile duct β-catenin synthesis orbile duct β-catenin phosphorylation and/or acetylation. In otherembodiments, the terms “treating” or “treatment” can refer to increasingthe amount of bile duct acetylated tubulin and/or bile duct β-cateninsynthesis.

As used herein, the term “polycystic disease” is a disease characterizedby the formation of cysts. Examples of polycystic disease includes, butis not limited to, renal cystic disease such as polycystic kidneydisease (PKD), polycystic liver disease (PLD), polycystic ovary syndrome(PCOS), pancreatic cysts, or combinations thereof.

Polycystic diseases can include cholangiopathies, a group of liverdiseases in which cholangiocytes, the epithelia lining the biliary treeare the target cells. “Cholangiopathies” include, but are not limitedto, primary biliary cirrhosis, primary sclerosing cholangitis, AIDScholangiopathy, disappearing bile duct syndromes, Alagille's syndrome,cystic fibrosis, and biliary atresia (see Tietz P S, Larusso N F (2006).“Cholangiocyte biology”. Current Opinion in Gastroenterology 22 (3):279-87).

Polycystic disease can also include liver diseases where the liver orliver function is compromised by dysfunction and/or imbalance in saltand/or water homeostasis or balance, such as diseases, disease states,and disorders that include the presence of liver cysts. Illustrativeliver diseases include, but are not limited to chronic hepatic liverfibrosis, PLD accompanying PKD, nephronophthisis (NPHP), Meckel-GruberSyndrome, Bardet-Biedl Syndrome, and the like.

A clinical symptom of polycystic liver disease that can be treated by anHDAC6 inhibitor compound of Formula I or II includes, but is not limitedto, hepatomegaly, multiple macrocysts within the liver, in cases ofisolated polycystic liver disease, or liver and kidney, in cases ofautosomal dominant polycystic kidney disease which can cause abdominaldistension, a shortness of breath, early postprandial fullness,abdominal discomfort, and back discomfort. Other clinical symptoms ofsevere polycystic liver disease include portal hypertension, varicealbleeding, jaundice, ascites and, in rare cases, cystic carcinoma as wellas “pseudo” Budd-Chiari syndrome resulting from a blockage of venousdrainage from the liver.

The present invention also includes pharmaceutically acceptable salts ofthe compounds described herein. As used herein, “pharmaceuticallyacceptable salts” refers to derivatives of the disclosed compoundswherein the parent compound is modified by converting an existing acidor base moiety to its salt form. Examples of pharmaceutically acceptablesalts include, but are not limited to, mineral or organic acid salts ofbasic residues such as amines; alkali or organic salts of acidicresidues such as carboxylic acids; and the like. The pharmaceuticallyacceptable salts of the present invention include the conventionalnon-toxic salts of the parent compound formed, for example, fromnon-toxic inorganic or organic acids. The pharmaceutically acceptablesalts of the present invention can be synthesized from the parentcompound which contains a basic or acidic moiety by conventionalchemical methods. Generally, such salts can be prepared by reacting thefree acid or base forms of these compounds with a stoichiometric amountof the appropriate base or acid in water or in an organic solvent, or ina mixture of the two; generally, nonaqueous media like ether, ethylacetate, ethanol, isopropanol, or acetonitrile are preferred. Lists ofsuitable salts are found in Remington's Pharmaceutical Sciences, 16thEdition, 1980 and 17th Edition, 1985, both published by Mack PublishingCompany, Easton, Pa., page 1418 and Journal of Pharmaceutical Science,66, 2 (1977), each of which is incorporated herein by reference in itsentirety.

Compounds of the Invention

In some embodiments, the HDAC6 specific inhibitor is a compound ofFormula I:

or a pharmaceutically acceptable salt thereof,

wherein,

ring B is aryl or heteroaryl;

R₁ is an aryl or heteroaryl, each of which may be optionally substitutedby OH, halo, or alkyl;

and

R is H or alkyl.

Representative compounds of Formula I are shown in TABLE I below andinclude, but are not limited to:

TABLE I

Compound A-1 2-(diphenylamino)-N-(7-(hydroxyamino)-7-oxoheptyl)pyrimidine-5-carboxamide IC₅₀(nM) HDAC6 = 10 HDAC3 = 84

Compound B-1 2-((2-chlorophenyl)(phenyl)amino)-N-(7-(hydroxyamino)-7-oxoheptyl)pyrimidine-5- carboxamide IC₅₀(nM) HDAC6 = 4HDAC3 = 76

or pharmaceutically acceptable salts thereof.

The preparation and properties of selective HDAC6 inhibitors accordingto Formula I are provided in International patent Application No.PCT/US2011/021982, the entire contents of which is incorporated hereinby reference.

In other embodiments, the HDAC6 specific inhibitor is a compound ofFormula II:

or a pharmaceutically acceptable salt thereof, wherein,

X is C or O;

R_(y) is independently, at each occurrence, selected from the groupconsisting of C₁₋₆-alkyl, C₁₋₆-alkoxy, halo, —C₁₋₆ haloalkyl, —C₁₋₆dihaloalkyl, —C₁₋₆ trihaloalkyl, —OH, —N(R¹)₂, —C(O)R′, —CO₂R¹, and—C(O)N(R¹)₂;

or:

two R_(y) groups on the same or adjacent carbon atoms are taken togetherto form a C₃₋₈-cycloalkyl or C₃₋₇-heterocycloalkyl ring, each of whichmay be fused or isolated;

R_(z) is independently, at each occurrence, selected from the groupconsisting of C₁₋₆-alkyl, C₁₋₆-alkoxy, halo, —C₁₋₆ haloalkyl, —C₁₋₆dihaloalkyl, —C₁₋₆ trihaloalkyl, —OH, —N(R²)₂, —C(O)R², —CO₂R²,—C(O)N(R²)₂,

each R¹ is independently, at each occurrence, selected from the groupconsisting of H, C₁₋₆-alkyl, C₃₋₈-cycloalkyl, C₃₋₇-heterocycloalkyl,aryl, heteroaryl, C₁₋₆-alkyl-cycloalkyl, C₁₋₆-alkyl-heterocycloalkyl,C₁₋₆-alkyl-aryl, and C₁₋₆-alkyl-heteroaryl;

each R² is independently, at each occurrence, selected from the groupconsisting of H or C₁₋₆-alkyl;

m is 0, 1, 2, or 3; and

n is 0, 1, 2, or 3.

In an embodiment of the compounds of Formula II, n is 1 or 2 and R_(z)is halo.

In another embodiment of the compounds of Formula II, X is C and m is 1or 2. In a preferred embodiment, X is C, m is 1 or 2, and R_(y) is haloor C₁₋₆-alkoxy. In another embodiment, two R_(y) groups on the same oradjacent carbon atoms are taken together to form a C₃₋₈-cycloalkyl orC₃₋₇-heterocycloalkyl ring and R_(y) is C₁₋₆-alkoxy. In a preferredembodiment, two R_(y) groups on the same carbon atom are taken togetherto form a C₃₋₈-cycloalkyl or C₃₋₇-heterocycloalkyl ring.

Preferred embodiments of Formula II, including pharmaceuticallyacceptable salts thereof, are shown below in TABLE II below. Allcompounds of Formula II, as well as pharmaceutically acceptable saltsthereof, and the compounds of TABLE II, as well as pharmaceuticallyacceptable salts thereof, are considered to be “compounds of theinvention.”

TABLE II Compound ID Structure A-2

B-2

C-2

D-2

The compositions and pharmaceutical compositions provided herein can beused to treat a subject's polycystic disease.

In certain embodiments, the polycystic disease is polycystic liverdisease.

In certain embodiments, the polycystic disease is renal cystic diseasesuch as polycystic kidney disease.

In certain embodiments, the compositions and pharmaceutical compositionsprovided herein can inhibit cholangiocyte proliferation and/or bile ductβ-catenin synthesis and/or induce bile duct β-catenin phosphorylationand/or acetylation.

In certain embodiments, the compositions and pharmaceutical compositionsprovided herein can increase the amount of bile duct acetylated tubulinand/or decrease bile duct β-catenin synthesis.

Another object of the present invention is the use of a compound asdescribed herein in the manufacture of a medicament for use in thetreatment of a polycystic disorder or disease herein. Another object ofthe present invention is the use of a compound as described herein foruse in the treatment of a polycystic disorder or disease herein.

Methods of the Invention

In one aspect of the invention, methods for the treatment of polycysticdisease are provided, comprising administering a therapeuticallyeffective amount of an HDAC6 specific inhibitor compound of Formula I orII, as described herein, to a subject in need thereof.

A therapeutically effective amount of an HDAC6 specific inhibitorcompound of Formula I or II sufficient to treat or ameliorate an effectof a polycystic disease may vary with the nature of the condition beingtreated, the length of time that activity is desired, and the age andthe condition of the patient, and ultimately may be determined by theattendant physician. The amount or dose of an HDAC6 specific inhibitorcompound of Formula I or II according to the present invention that maybe administered to a patient may also vary depending on a variety offactors known in the art e.g., species, sex, age, weight, condition ofthe patient, desired response, nature of the condition, metabolism,severity of disease, side-effects. The desired dose may be convenientlyadministered in a single dose, or as multiple doses administered atappropriate intervals, for example as two, three, four or more sub-dosesper day. Multiple doses often are desired, or required.

In certain embodiments, the polycystic disease can be a polycystic liverdisease (PLD). PLDs are genetic disorders that can be included in thecholangiopathies, a group of diseases of diverse etiologies all of whichhave the cholangiocyte as the target cell. PLDs occur either alone ortogether with polycystic kidney disease (PKD) (see Masyuk et al.Cholangiociliopathies: genetics, molecular mechanisms and potentialtherapies, Current Opinion in Gastroenterology (2009) 25:265-271).

In certain embodiments, the subject being treated has one or moremutations in at least one of the PRKCSH (Protein Kinase C Substrate80K-H) and Sec63 genes. These mutations cause polycystic liver diseasewithout kidney involvement (ADPLD) by affecting the cell'sposttranslational protein modification machinery and ciliary signaltransduction via polycystin-2 degradation ((see Drenth et al. Germlinemutations in PRKCSH are associated with autosomal dominant polycysticliver disease, Nature Genetics (2003) 33:345-347 3; Davila et al.Mutations in SEC63 cause autosomal dominant polycystic liver disease,Nat Genet (2004) 36:575-577 4; Woollattet al. Human Sec63 endoplasmicreticulum membrane protein, map position 6q21, Chromosome Research(1999), 7:77; Gao et al. PRKCSH/80K-H, the protein mutated in polycysticliver disease, protects polycystin-2/TRPP2 against HERP-mediateddegradation, Human Molecular Genetics (2010) 19:16-24).

In certain embodiments, the subject being treated has one or moremutations in at least on of the Pkd1 and Pkd2 genes, which encode theciliary-associated proteins, polycystin-1 (PC1) and -2 (PC2), and whichare causative for cystic degeneration of the liver and kidneys inautosomal-dominant polycystic kidney disease (ADPKD).

In certain embodiments, the subject being treated has one or moremutations in the Pkhd1 gene which is associated with autosomal-recessivepolycystic kidney disease (ARPKD) (see Hughes et al. The polycystickidney disease 1 (PKD1) gene encodes a novel protein with multiple cellrecognition domains, Nature Genetics (1995) 10:151-160; Mochizuki et al.PKD2, a gene for polycystic kidney disease that encodes an integralmembrane protein, Science (1996) 272:1339-13428; Onuchic et al. PKHD1,the polycystic kidney and hepatic disease 1 gene, encodes a novel largeprotein containing multiple immunoglobulin-likeplexin-transcription-factor domains and parallel beta-helix 1 repeats,Am J Hum Genet (2002)70:1305-1317; Ward et al. The gene mutated inautosomal recessive polycystic kidney disease encodes a large,receptor-like protein, Nat Genet (2002) 30:259-269).

As used herein, the term “renal cystic disease” includes, but not belimited to, a large group of diseases, including polycystic kidneydisease (PCK), von Hippel-Lindau, tuberosclerosis, nephronophthisis,autosomal dominant polycystic kidney disease (ADPKD), autosomalrecessive polycystic kidney disease (ARPKD), acquired cystic kidneydisease (ACKD), and autosomal dominant polycystic liver disease (ARPKD)(see, for example, Friedman, J. Cystic Diseases of the Kidney, inPRINCIPLES AND PRACTICE OF MEDICAL GENETICS (A. Emery and D. Rimoin,Eds.) pp. 1002-1010, Churchill Livingston, Edinburgh, U. K. (1983);Striker & Striker (1986) Am. J. Nephrol. 6:161-164. Extrarenalmanifestations include hepatic and pancreatic cysts as well ascardiovascular complications. Gabow & Grantham (1997) Polycystic KidneyDisease, in DISEASES OF THE KIDNEY (R. Schrier & C. Gottschalk, Eds.),pp. 521-560, Little Brown, Boston; Welling & Grantham (1996) CysticDiseases of the Kidney, in RENAL PATHOLOGY (C. Tisch & B. Brenner, Eds.)pp:1828-1863, Lippincott, Philadelphia).

In another aspect, a therapeutically effective amount of a compound ofFormula I or II is injected intraperitoneally into a subject with apolycystic disease, wherein the compound of Formula I or II treats thepolycystic disease.

In certain embodiments, the HDAC6-specific inhibitor compound of FormulaI or II prevents cyst formation.

In certain embodiments, the HDAC6-specific inhibitor compound of FormulaI or II inhibits cyst growth.

In certain embodiments, the HDAC6-specific inhibitor compound of FormulaI or II inhibits cholangiocyte proliferation.

HDAC6 inhibition with a compound of Formula I or II can decrease manyhallmarks of polycystic liver disease including hepatomegaly and thegrowth of cysts in the liver or kidney.

In certain embodiments, HDAC6 inhibition with a compound of Formula I orII prevents the formation of cysts in patients that are geneticallypre-disposed to acquiring a polycystic disease.

In a preferred embodiment, the compound of Formula I can be CompoundA-1.

In a preferred embodiment, the compound of Formula I can be CompoundB-1.

In a preferred embodiment, the compound of Formula II can be CompoundC-2.

In certain embodiments, pharmacological inhibition of HDAC6 with acompound of Formula I or II treats polycystic disease by inhibitingβ-catenin function e.g. inhibiting the expression of and nuclearaccumulation of β-catenin.

In one aspect of the invention, a method for the treatment of polycysticliver disease is provided comprising administering to a subject withpolycystic liver disease a therapeutically effective amount of the HDAC6specific inhibitor compound (Compound A-1) having the structure of:

or pharmaceutically acceptable salts thereof,

wherein the amount of the HDAC6 specific inhibitor compound A-1 iseffective at inhibiting cyst growth.

In one aspect of the invention, a method for the treatment of polycysticliver disease is provided comprising administering to a subject withpolycystic liver disease a therapeutically effective amount of the HDAC6specific inhibitor compound B-1 having the structure of:

or pharmaceutically acceptable salts thereof,

wherein the amount of the HDAC6 specific inhibitor compound B-1 iseffective at inhibiting cyst growth.

In one aspect of the invention, a method for the treatment of polycysticliver disease is provided comprising administering to a subject withpolycystic liver disease a therapeutically effective amount of the HDAC6specific inhibitor compound C-2 having the structure of:

or pharmaceutically acceptable salts thereof, wherein the amount of theHDAC6 specific inhibitor compound C-2 is effective at inhibiting cystgrowth.

Pharmaceutical Compositions

In another aspect, the invention provides a pharmaceutical compositioncomprising a compound of Formula I or II, or a pharmaceuticallyacceptable ester, salt, or pro-drug thereof, together with apharmaceutically acceptable carrier. This pharmaceutical composition canbe used in the treatment of polycystic diseases.

Compounds of the invention can be administered as pharmaceuticalcompositions by any conventional route, in particular enterally, e.g.,orally, e.g., in the form of tablets or capsules, or parenterally, e.g.,in the form of injectable solutions or suspensions, topically, e.g., inthe form of lotions, gels, ointments or creams, or in a nasal orsuppository form. Pharmaceutical compositions comprising a compound ofthe present invention in free form or in a pharmaceutically acceptablesalt form in association with at least one pharmaceutically acceptablecarrier or diluent can be manufactured in a conventional manner bymixing, granulating or coating methods. For example, oral compositionscan be tablets or gelatin capsules comprising the active ingredienttogether with a) diluents, e.g., lactose, dextrose, sucrose, mannitol,sorbitol, cellulose and/or glycine; b) lubricants, e.g., silica, talcum,stearic acid, its magnesium or calcium salt and/or polyethyleneglycol;for tablets also c) binders, e.g., magnesium aluminum silicate, starchpaste, gelatin, tragacanth, methylcellulose, sodiumcarboxymethylcellulose and or polyvinylpyrrolidone; if desired d)disintegrants, e.g., starches, agar, alginic acid or its sodium salt, oreffervescent mixtures; and/or e) absorbents, colorants, flavors andsweeteners. Injectable compositions can be aqueous isotonic solutions orsuspensions, and suppositories can be prepared from fatty emulsions orsuspensions. The compositions may be sterilized and/or containadjuvants, such as preserving, stabilizing, wetting or emulsifyingagents, solution promoters, salts for regulating the osmotic pressureand/or buffers. In addition, they may also contain other therapeuticallyvaluable substances. Suitable formulations for transdermal applicationsinclude an effective amount of a compound of the present invention witha carrier. A carrier can include absorbable pharmacologically acceptablesolvents to assist passage through the skin of the host. For example,transdermal devices are in the form of a bandage comprising a backingmember, a reservoir containing the compound optionally with carriers,optionally a rate controlling barrier to deliver the compound to theskin of the host at a controlled and predetermined rate over a prolongedperiod of time, and means to secure the device to the skin. Matrixtransdermal formulations may also be used. Suitable formulations fortopical application, e.g., to the skin and eyes, are preferably aqueoussolutions, ointments, creams or gels well-known in the art. Such maycontain solubilizers, stabilizers, tonicity enhancing agents, buffersand preservatives.

It will also be appreciated that the compounds and pharmaceuticalcompositions of the present invention can be formulated and employed incombination therapies, that is, the compounds and pharmaceuticalcompositions can be formulated with or administered concurrently with,prior to, or subsequent to, one or more other desired therapeutics ormedical procedures. The particular combination of therapies(therapeutics or procedures) to employ in a combination regimen willtake into account compatibility of the desired therapeutics and/orprocedures and the desired therapeutic effect to be achieved. It willalso be appreciated that the therapies employed may achieve a desiredeffect for the same disorder (for example, an inventive compound may beadministered concurrently with another immunomodulatory agent,anticancer agent or agent useful for the treatment of an autoimmunedisease such as SLE), or they may achieve different effects (e.g.,control of any adverse effects).

In certain embodiments, the pharmaceutical compositions of the presentinvention further comprise one or more additional therapeutically activeingredients. For purposes of the invention, the term “palliative” refersto treatment that is focused on the relief of symptoms of a diseaseand/or side effects of a therapeutic regimen, but is not curative. Forexample, palliative treatment encompasses painkillers, anti-nauseamedications, anti-pyretics, and anti-sickness drugs. In addition,chemotherapy, radiotherapy and surgery can all be used palliatively(that is, to reduce symptoms without going for cure; e.g., for shrinkingtumors and reducing pressure, bleeding, pain and other symptoms ofcancer).

The pharmaceutical compositions of the present invention comprise atherapeutically effective amount of a compound of the present inventionformulated together with one or more pharmaceutically acceptablecarriers.

As used herein, the term “pharmaceutically acceptable carrier” means anon-toxic, inert solid, semi-solid or liquid filler, diluent,encapsulating material or formulation auxiliary of any type. Thepharmaceutical compositions of this invention can be administered tohumans and other animals orally, rectally, parenterally,intracisternally, intravaginally, intraperitoneally, topically (as bypowders, ointments, or drops), buccally, or as an oral or nasal spray.

Liquid dosage forms for oral administration include pharmaceuticallyacceptable emulsions, microemulsions, solutions, suspensions, syrups andelixirs. In addition to the active compounds, the liquid dosage formsmay contain inert diluents commonly used in the art such as, forexample, water or other solvents, solubilizing agents and emulsifierssuch as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethylacetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butyleneglycol, dimethylformamide, oils (in particular, cottonseed, groundnut,corn, germ, olive, castor, and sesame oils), glycerol,tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid estersof sorbitan, and mixtures thereof. Besides inert diluents, the oralcompositions can also include adjuvants such as wetting agents,emulsifying and suspending agents, sweetening, flavoring, and perfumingagents.

Injectable preparations, for example, sterile injectable aqueous oroleaginous suspensions may be formulated according to the known artusing suitable dispersing or wetting agents and suspending agents. Thesterile injectable preparation may also be a sterile injectablesolution, suspension or emulsion in a nontoxic parenterally acceptablediluent or solvent, for example, as a solution in 1,3-butanediol. Amongthe acceptable vehicles and solvents that may be employed are water,Ringer's solution, U.S.P. and isotonic sodium chloride solution. Inaddition, sterile, fixed oils are conventionally employed as a solventor suspending medium. For this purpose any bland fixed oil can beemployed including synthetic mono- or diglycerides. In addition, fattyacids such as oleic acid are used in the preparation of injectables.

According to the methods of treatment of the present invention,disorders are treated or prevented in a subject, such as a human orother animal, by administering to the subject a therapeuticallyeffective amount of a compound of the invention, in such amounts and forsuch time as is necessary to achieve the desired result.

The term “therapeutically effective amount” of a compound of theinvention, as used herein, means a sufficient amount of the compound soas to decrease one or more of the symptoms caused by a polycystic liverdisease in a subject.

As is well understood in the medical arts a therapeutically effectiveamount of a compound of this invention will be at a reasonablebenefit/risk ratio applicable to any medical treatment.

In general, compounds of the invention will be administered intherapeutically effective amounts via any of the usual and acceptablemodes known in the art, either singly or in combination with one or moretherapeutic agents. A therapeutically effective amount may vary widelydepending on the severity of the disease, the age and relative health ofthe subject, the potency of the compound used and other factors. Ingeneral, satisfactory results are indicated to be obtained systemicallyat daily dosages of from about 0.03 to 2.5 mg/kg per body weight (0.05to 4.5 mg/m²). An indicated daily dosage in the larger mammal, e.g.humans, is in the range from about 0.5 mg to about 100 mg, convenientlyadministered, e.g. in divided doses up to four times a day or incontrolled release form. Suitable unit dosage forms for oraladministration comprise from ca. 1 to 50 mg active ingredient.

In certain embodiments, a therapeutic amount or dose of the compounds ofthe present invention may range from about 0.1 mg/kg to about 500 mg/kg(about 0.18 mg/m² to about 900 mg/m²), alternatively from about 1 toabout 50 mg/kg (about 1.8 to about 90 mg/m²). In general, treatmentregimens according to the present invention comprise administration to apatient in need of such treatment from about 10 mg to about 1000 mg ofthe compound(s) of this invention per day in single or multiple doses.Therapeutic amounts or doses will also vary depending on route ofadministration, as well as the possibility of co-usage with otheragents.

Upon improvement of a subject's condition, a maintenance dose of acompound, composition or combination of this invention may beadministered, if necessary. Subsequently, the dosage or frequency ofadministration, or both, may be reduced, as a function of the symptoms,to a level at which the improved condition is retained when the symptomshave been alleviated to the desired level, treatment should cease. Thesubject may, however, require intermittent treatment on a long-termbasis upon any recurrence of symptoms resulting from the polycysticliver disease.

It will be understood, however, that the total daily usage of thecompounds and compositions of the present invention will be decided bythe attending physician within the scope of sound medical judgment. Thespecific inhibitory dose for any particular patient will depend upon avariety of factors including the disorder being treated and the severityof the disorder; the activity of the specific compound employed; thespecific composition employed; the age, body weight, general health, sexand diet of the patient; the time of administration, route ofadministration, and rate of excretion of the specific compound employed;the duration of the treatment; drugs used in combination or coincidentalwith the specific compound employed; and like factors well known in themedical arts.

Kits

The disclosure herein provides for a kit format which comprises packageunits having different doses of the compound of Formula I or II fortreating a polycystic disease in a subject. In certain embodiments, thecompound of Formula I is the compound A-1 or compound B-1. In otherembodiments, the compound of Formula II is the compound C-2.

The kit may also contain one or more of the following items:instructions for use including prescribing information, dosageinformation, storage information, and the like as well as sterile salinesolution, needles, syringes, catheters and first aid materials such asbandages etc. Kits may include containers of reagents mixed together insuitable proportions for performing the methods described herein.Reagent containers preferably contain reagents in unit quantities thatobviate measuring steps when performing the subject methods.

The package label can include, for example, instructions to take thecompound of Formula I or II for the treatment of a polycystic disease.In another embodiment, the package label includes instructions to treatpolycystic liver disease.

Packaged compositions are also provided that comprise a therapeuticallyeffective amount of a compound of Formula I or Formula II, and apharmaceutically acceptable carrier or diluent as well as instructionson how to treat a polycystic disease such as polycystic liver disease.

Compounds of the present invention can be conveniently prepared orformed during the process of the invention, as solvates (e.g.,hydrates). Hydrates of compounds of the present invention can beconveniently prepared by recrystallization from an aqueous/organicsolvent mixture, using organic solvents such as dioxan, tetrahydrofuranor methanol.

In addition, some of the compounds of this invention have one or moredouble bonds, or one or more asymmetric centers. Such compounds canoccur as racemates, racemic mixtures, single enantiomers, individualdiastereomers, diastereomeric mixtures, and cis- or trans- or E- orZ-double isomeric forms, and other stereoisomeric forms that may bedefined, in terms of absolute stereochemistry, as (R)- or (S)-, or as(D)- or (L)- for amino acids. All such isomeric forms of these compoundsare expressly included in the present invention. Optical isomers may beprepared from their respective optically active precursors by theprocedures described above, or by resolving the racemic mixtures. Theresolution can be carried out in the presence of a resolving agent, bychromatography or by repeated crystallization or by some combination ofthese techniques which are known to those skilled in the art. Furtherdetails regarding resolutions can be found in Jacques, et al.,Enantiomers, Racemates, and Resolutions (John Wiley & Sons, 1981). Thecompounds of this invention may also be represented in multipletautomeric forms, in such instances, the invention expressly includesall tautomeric forms of the compounds described herein. When thecompounds described herein contain olefinic double bonds or othercenters of geometric asymmetry, and unless specified otherwise, it isintended that the compounds include both E and Z geometric isomers.Likewise, all tautomeric forms are also intended to be included. Theconfiguration of any carbon-carbon double bond appearing herein isselected for convenience only and is not intended to designate aparticular configuration unless the text so states; thus a carbon-carbondouble bond depicted arbitrarily herein as trans may be cis, trans, or amixture of the two in any proportion. All such isomeric forms of suchcompounds are expressly included in the present invention. All crystalforms of the compounds described herein are expressly included in thepresent invention.

The synthesized compounds can be separated from a reaction mixture andfurther purified by a method such as column chromatography, highpressure liquid chromatography, or recrystallization. As can beappreciated by the skilled artisan, further methods of synthesizing thecompounds of the formulae herein will be evident to those of ordinaryskill in the art. Additionally, the various synthetic steps may beperformed in an alternate sequence or order to give the desiredcompounds. In addition, the solvents, temperatures, reaction durations,etc. delineated herein are for purposes of illustration only and one ofordinary skill in the art will recognize that variation of the reactionconditions can produce the desired compounds of the present invention.Synthetic chemistry transformations and protecting group methodologies(protection and deprotection) useful in synthesizing the compoundsdescribed herein are known in the art and include, for example, thosesuch as described in R. Larock, Comprehensive Organic Transformations,VCH Publishers (1989); T. W. Greene and P.G.M. Wuts, Protective Groupsin Organic Synthesis, 2d. Ed., John Wiley and Sons (1991); L. Fieser andM. Fieser, Fieser and Fieser's Reagents for Organic Synthesis, JohnWiley and Sons (1994); and L. Paquette, ed., Encyclopedia of Reagentsfor Organic Synthesis, John Wiley and Sons (1995), and subsequenteditions thereof.

The compounds of this invention may be modified by appending variousfunctionalities via any synthetic means delineated herein to enhanceselective biological properties. Such modifications are known in the artand include those which increase biological penetration into a givenbiological system (e.g., blood, lymphatic system, central nervoussystem), increase oral availability, increase solubility to allowadministration by injection, alter metabolism and alter rate ofexcretion.

The compounds of the invention are defined herein by their chemicalstructures and/or chemical names. Where a compound is referred to byboth a chemical structure and a chemical name, and the chemicalstructure and chemical name conflict, the chemical structure isdeterminative of the compound's identity.

The recitation of a listing of chemical groups in any definition of avariable herein includes definitions of that variable as any singlegroup or combination of listed groups. The recitation of an embodimentherein includes that embodiment as any single embodiment or incombination with any other embodiments or portions thereof. Therecitation of an embodiment for a variable herein includes thatembodiment as any single embodiment or in combination with any otherembodiments or portions thereof.

Any patent, patent application, publication, or other disclosurematerial identified in the specification is hereby incorporated byreference herein in its entirety. Any material, or portion thereof, thatis said to be incorporated by reference herein, but which conflicts withexisting definitions, statements, or other disclosure material set forthherein is only incorporated to the extent that no conflict arisesbetween that incorporated material and the present disclosure material.

EXAMPLES

Examples have been set forth below for the purpose of illustration andto describe certain specific embodiments of the invention. However, thescope of the claims is not to be in any way limited by the examples setforth herein. Various changes and modifications to the disclosedembodiments will be apparent to those skilled in the art and suchchanges and modifications including, without limitation, those relatingto the chemical structures, subtitutents, derivatives, formulationsand/or methods of the invention may be made without departing from thespirit of the invention and the scope of the appended claims.Definitions of the variables in the structures in the schemes herein arecommensurate with those of corresponding positions in the formulaepresented herein.

Example 1: Synthesis of the Compounds of the Invention

The synthesis of the compounds of Formula I is provided inPCT/US2011/021982, which is incorporated herein by reference in itsentirety.

Synthesis of Compound A-1

Reaction Scheme

Synthesis of Intermediate 2

A mixture of aniline (3.7 g, 40 mmol), ethyl2-chloropyrimidine-5-carboxylate 1 (7.5 g, 40 mmol), K₂CO₃ (11 g, 80mmol) in DMF (100 ml) was degassed and stirred at 120° C. under N₂overnight. The reaction mixture was cooled to rt and diluted with EtOAc(200 ml), then washed with saturated brine (200 ml×3). The organic layerwas separated and dried over Na₂SO₄, evaporated to dryness and purifiedby silica gel chromatography (petroleum ethers/EtOAc=10/1) to give thedesired product as a white solid (6.2 g, 64%).

Synthesis of Intermediate 3

A mixture of the compound 2 (6.2 g, 25 mmol), iodobenzene (6.12 g, 30mmol), CuI (955 mg, 5.0 mmol), Cs₂CO₃ (16.3 g, 50 mmol) in TEOS (200 ml)was degassed and purged with nitrogen. The resulting mixture was stirredat 140° C. for 14 h. After cooling to r.t., the residue was diluted withEtOAc (200 ml) and 95% EtOH (200 ml), NH₄F—H₂O on silica gel [50 g,pre-prepared by the addition of NH₄F (100 g) in water (1500 ml) tosilica gel (500 g, 100-200 mesh)] was added, and the resulting mixturewas kept at r.t. for 2 h, the solidified materials was filtered andwashed with EtOAc. The filtrate was evaporated to dryness and theresidue was purified by silica gel chromatography (petroleumethers/EtOAc=10/1) to give a yellow solid (3 g, 38%).

Synthesis of Intermediate 4

2N NaOH (200 ml) was added to a solution of the compound 3 (3.0 g, 9.4mmol) in EtOH (200 ml). The mixture was stirred at 60° C. for 30 min.After evaporation of the solvent, the solution was neutralized with 2NHCl to give a white precipitate. The suspension was extracted with EtOAc(2×200 ml), and the organic layer was separated, washed with water(2×100 ml), brine (2×100 ml), and dried over Na₂SO₄. Removal of solventgave a brown solid (2.5 g, 92%).

Synthesis of Intermediate 6

A mixture of compound 4 (2.5 g, 8.58 mmol), aminoheptanoate 5 (2.52 g,12.87 mmol), HATU (3.91 g, 10.30 mmol), DIPEA (4.43 g, 34.32 mmol) wasstirred at r.t. overnight. After the reaction mixture was filtered, thefiltrate was evaporated to dryness and the residue was purified bysilica gel chromatography (petroleum ethers/EtOAc=2/1) to give a brownsolid (2 g, 54%).

Synthesis of2-(diphenylamino)-N-(7-(hydroxyamino)-7-oxoheptyl)pyrimidine-5-carboxamide

A mixture of the compound 6 (2.0 g, 4.6 mmol), sodium hydroxide (2N, 20mL) in MeOH (50 ml) and DCM (25 ml) was stirred at 0° C. for 10 min.Hydroxylamine (50%) (10 ml) was cooled to 0° C. and added to themixture. The resulting mixture was stirred at r.t. for 20 min. Afterremoval of the solvent, the mixture was neutralized with 1M HCl to givea white precipitate. The crude product was filtered and purified bypre-HPLC to give a white solid (950 mg, 48%).

Synthesis of Compound B-1

Reaction Scheme:

Synthesis of Intermediate 2: See synthesis of intermediate 2 insynthesis of Compound A-1 above.

Synthesis of Intermediate 3

A mixture of compound 2 (69.2 g, 1 equiv.), 1-chloro-2-iodobenzene(135.7 g, 2 equiv.), Li₂CO₃ (42.04 g, 2 equiv.), K₂CO₃ (39.32 g, 1equiv.), Cu (1 equiv. 45 μm) in DMSO (690 ml) was degassed and purgedwith nitrogen. The resulting mixture was stirred at 140° C. Work-up ofthe reaction gave compound 3 at 93% yield.

Synthesis of Intermediate 4: See synthesis of intermediate 4 insynthesis of Compound A-1 above.

Synthesis of Intermediate 6: See synthesis of intermediate 6 insynthesis of Compound A-1 above.

Synthesis of2-((2-chlorophenyl)(phenyl)amino)-N-(7-(hydroxyamino)-7-oxoheptyl)pyrimidine-5-carboxamide(Compound B-1)

See synthesis of Compound A-1 above.

Another embodiment is a method of making a compound of Formula I usingany one, or combination of, reactions delineated herein. The method caninclude the use of one or more intermediates or chemical reagentsdelineated herein.

Synthesis of Compound C-2

Step 1: To a solution of 1 (2.00 g, 12.81 mmol) and 2 (1.552 g, 12.81mmol) in THF (20 mL) was added Ti(OEt)₄ (5.4 mL, 25.56 mmol). Themixture was stirred at r.t. for 16 hrs and then poured into saturatedNaHCO₃ solution at 0° C. The resulting precipitate was filtered off. Theresulting filtrate was extracted with EA. The combined EA layers wereconcentrated in vacuo and the residue was purified by silica gelchromatography (PE/EA=4/1, 2/1) to afford 3 as a white solid (2.61 g,yield: 75%).

Step 2: To a flask containing 3 (1.00 g, 3.86 mmol) was added a solutionof PhMgBr (1M in THF, 10 mL) at 0° C. It was stirred at 0° C. to rtuntil a complete reaction. Saturated NH₄Cl solution was added to adjustpH 6-7. The resulting mixture was extracted with EA. The combined EAlayers were concentrated in vacuo and the residue was purified by silicagel chromatography (PE/EA=5/1, 2/1, 1.5/1) to afford 4 as a white solid(823 mg, yield: 60%).

Step 3: A mixture of compound 4 (8.3 mg, 2.38 mmol) in HCl (2M in water,20 mL) and THF (20 mL) was stirred at 50° C. for 16 hrs. A solution ofNaOH was added to the mixture to adjust pH 7-8. THF was removed in vacuoand the aqueous phase was extracted with EA. The combined EA layers wereconcentrated in vacuo and the residue was dissolved in EA. HCl (4 M, 1mL) was added. The resulting white solid was collected by filtration toafford desired product 5 (395 mg, yield: 57%).

Step 4: A mixture of compound 5 (350 mg, 1.55 mmol), 6 (376 mg, 2.02mol), and DIPEA (1.07 mL, 6.47 mmol) in NMP (4 mL) was stirred at 130°C. for 5 hrs. The mixture was added water (20 mL), extracted with EA (25mL×2). The organic layer was concentrated to get a residue, which waspurified by silica gel chromatography (PE/EA=4/1) to afford 7 (178 mg,yield: 34%).

Step 5: To a solution of compound 7 (168 mg, 0.50 mmol) in DCM (30 mL)was added DAST (302 μL, 2.47 mmol) at 0° C. It was stirred at rt for 3hrs and 35° C. for 2 hrs. The reaction mixture was quenched withsaturated NaHCO₃ (5 mL), and extracted with EtOAc (2×5 mL). The organicextracts were concentrated in vacuo. The residue was purified by pre-TLCto give 8 (74 mg, yield: 42%).

Step 6: NH₂OH (50% in water, 3.9 mL) was added to a flask containing 8(74 mg, 0.20 mmol) at 0° C. Then saturated NaOH solution in MeOH (3.9ml) was added at 0° C. DCM (3.9 mL) was added to aid substrate todissolve. The mixture was heated at 25° C. for 18 hrs. Con. HCl wasadded to adjust pH to 7. It was concentrated in vacuo and the residuewas purified by pre-HPLC to afford Compound C-2 (27 mg, yield: 38%) as awhite solid. ¹H NMR (500 MHz, DMSO) δ 10.96 (s, 1H), 9.00 (s, 1H), 8.63(s, 1H), 8.35 (s, 1H), 8.29 (s, 1H), 7.39 (d, J=7.6 Hz, 2H), 7.28 (t,J=7.7 Hz, 2H), 7.17 (t, J=7.3 Hz, 1H), 2.73 (s, 2H), 2.23-1.88 (m, 6H).LCMS: m/z=349 (M+H)⁺.

Example 2: HDAC Enzyme Assays

Compounds for testing were diluted in DMSO to 50 fold the finalconcentration and a ten point three fold dilution series was made. Thecompounds were diluted in assay buffer (50 mM HEPES, pH 7.4, 100 mM KCl,0.001% Tween-20, 0.05% BSA, 20 μM TCEP) to 6 fold their finalconcentration. The HDAC enzymes (purchased from BPS Biosciences) werediluted to 1.5 fold their final concentration in assay buffer. Thetripeptide substrate and trypsin at 0.05 μM final concentration werediluted in assay buffer at 6 fold their final concentration. The finalenzyme concentrations used in these assays were 3.3 ng/ml (HDAC1), 0.2ng/ml (HDAC2), 0.08 ng/ml (HDAC3) and 2 ng/ml (HDAC6). The finalsubstrate concentrations used were 16 μM (HDAC1), 10 μM (HDAC2), 17 μM(HDAC3) and 14 μM (HDAC6).

Five μ1 of compounds and 20 μl of enzyme were added to wells of a black,opaque 384 well plate in duplicate. Enzyme and compound were incubatedtogether at room temperature for 10 minutes. Five μ1 of substrate wasadded to each well, the plate was shaken for 60 seconds and placed intoa Victor 2 microtiter plate reader. The development of fluorescence wasmonitored for 60 min and the linear rate of the reaction was calculated.The IC50 was determined using Graph Pad Prism by a four parameter curvefit. The IC50 values (nM) obtained for the compound C-2 can be found inTABLE III, below.

TABLE III Compound ID Structure HDAC1 HDAC2 HDAC3 HDAC6 C-2

961 982 4380 3.7

The IC50 values (nM) obtained for compounds A-1 and B-1 can be found inTABLE I.

Example 3: HDAC6 is Overexpressed in Cystic Cholangiocytes PolymeraseChain Reaction

RNA was isolated from control and PCK rat cholangiocytes with TRIZOLreagent (Invitrogen). cDNA was obtained using First Srand cDNA Synthesis(Invitrogen) reagents and HDAC6 primers (forward primer: 5′-TCA GCG CAGTCT TAT GGA TG-3′ (SEQ ID NO.: 1); reverse primer: 5′-GCG GTG GAT GGAGAA ATA GA-3′, SEQ ID NO.: 2) were purchased from Integrated DNATechnologies. β-catenin mRNA expression was analyzed using the TaqManGene Expression Assay (Assay ID Rn00584431_g1) following themanufacturer directions. The samples were normalized to 18S rRNA.

Western Blots

Protein isolated from cultured control rat and PCK rat cholangiocyteswere analyzed. Cells were scrapped, resuspended in Phosphate BufferedSaline (PBS) with Protease Inhibitors, Sodium Orthovanadate andPhenylmethanesulfonylfluoride (PMSF), sonicated and equal amounts ofsample protein were diluted in Laemmli Sample Buffer (Biorad) andmercaptoethanol. After SDS polyacrylamide gel electrophoresis, SDS-PAGE,proteins were transferred to nitrocellulose membranes, blocked, and thenincubated with the following primary antibodies: HDAC6 (Santa CruzBiotechnology, D-11, 1:500), acetylated-α-tubulin (Sigma Aldrich,1:2000), β-catenin (Cell Signaling Technology, (D10A8) XP® Rabbit mAb;1:1000), Phospho-β-Catenin (Ser33/37/Thr41) (Cell Signaling Technology,1:1000), acetyl-β-catenin (Cell Signaling Technology, 1:1000), c-myc(Santa Cruz Biotechnology, 1:500), cyclin D1 (Santa Cruz Biotechnology,1:500), and actin (Sigma Aldrich, 1:5000). The membranes were incubatedovernight at 4° C., washed and incubated for 1 hour at room temperaturewith horseradish perodixase-conjugated (1:5000, Invitrogen) or IRdye 680or 800 (1:15000, Odyssey) corresponding secondary antibody. The ECLsystem or Odyssey Licor Scanner was used for protein detection and theGel-Pro Analyzer 6.0 software was used for densitometry analysis.

Immunofluorescence and Confocal Microscopy.

Paraffin-embedded liver sections of control and PCK rats, healthy humanbeings, and ADPKD and ARPKD patients were incubated with antibodiesagainst HDAC6 (Santa Cruz Biotechnology, D-11, 1:100) andacetylated-α-tubulin (Sigma Aldrich, mouse monoclonal 1:500) overnightat 4° C. followed by 90 minutes at room temperature of fluorescentsecondary antibody incubation. Nuclei were stained with DAPI (ProLongGold Antifade Reagent, Life Technologies). Images were acquired with aZeiss LSM 510 confocal microscope. Paraffin blocks from 3 normal, 3ARPKD, and 3 ADPKD patients were obtained from the Mayo Clinic TissueRegistry Archives. All experimental procedures were approved by the MayoClinic Institutional Review Boards (IRB #: 08-005681).

Statistics

Data are expressed as mean±SE. Statistical analyses were conducted bytwo-tailed; Student's t-tests were used to compare two groups. Thecut-off p-value for significance was set at p<0.05.

To assess the expression of HDAC6 mRNA in cholangiocytes, mRNA isolatedfrom cultured rat cholangiocytes was analyzed using PCR. As shown inFIG. 1A, HDAC6 mRNA is present in both control and PCK ratcholangiocytes. The identity of the PCR product was confirmed bysequencing.

FIG. 1B shows a Western blot analysis on proteins isolated from culturedcontrol rat and PCK rat cholangiocytes. HDAC6 protein is overexpressed6.5-fold in PCK cholangiocytes compared to control rat cholangiocytes,which correlates with decreased levels of acetylated-α-tubulin, one ofthe principal HDAC6 substrates (see Zhang et al. Two catalytic domainsare required for protein deacetylation, J. Biol. Chem. (2006)281:2401-2404).

The overexpression of HDAC6 observed in vitro was also confirmed in vivoby confocal immunofluorescence of liver tissues. In liver tissues of PCKrats, as well as ADPKD and ARPKD patients, the immunoreactivity of HDAC6is increased in hepatic cysts compared to normal rats and healthy humancontrols, respectively (FIG. 1C).

Example 4: Inhibition of HDAC6 Decreases Cholangiocyte Proliferation andCysts Growth In Vitro Tissue Culture

For in vitro experiments, cholangiocytes were isolated from controlrats, PCK rats (an animal model for ARPKD; Vroman B, LaRusso N F:Development and characterization of polarized primary cultures of ratintrahepatic bile duct epithelial cells, Lab Invest 1996, 74:303-313),healthy human beings and ADPKD patients (Masyuk et al. Biliary exosomesinfluence cholangiocyte regulatory mechanisms and proliferation throughinteraction with primary cilia, Am J Physiol Gastrointest Liver Physiol(2010) 299:G990-999; O'Haraet al. Cholangiocyte N-Ras protein mediateslipopolysaccharide-induced interleukin 6 secretion and proliferation, JBiol Chem (2011) 286:30352-30360; Banaleset al. Up-regulation ofmicroRNA 506 leads to decreased Cl(−)/HCO(3) (−) anion exchanger 2expression in biliary epithelium of patients with primary biliarycirrhosis, Hepatology (2012) 56(2):687-97).

Cholangiocytes were cultured in Collagen-I-coated flasks (BD Biocoat).All cell lines were incubated in NRC Media at 37° C., 5% CO2, 100%humidity. NRC Media contains Dulbecco's modified Eagle medium/F12 withthe following additions: 0.01 mL/mL Minimum Essential Media (MEM)nonessential amino acids, 0.01 mL/mL lipid concentrate, 0.01 mL/mL MEMvitamin solution, 2 mmol/L L-glutamine, 0.05 mg/mL soybean trypsininhibitor, 0.01 mL/mL insulin/transferring/selenium-S, 5% fetal bovineserum, 30 g/mL bovine pituitary extract, 25 ng/mL epidermal growthfactor, 393 ng/mL dexamethasone, 3.4 g/mL 3,3-,5-triiodo-L-thyronine,4.11 g/mL forskolin, and 1% penicillin-streptomycin.

Proliferation Assays

Control and PCK rat cholangiocytes were cultured on Collagen-I-coatedflasks (BD Biocoat) using NRC Media, detached with 0.25% Trypsin-EDTA(GIBCO), transferred to Collagen-I-coated 96-well plates (10 000cells/well) and incubated at 37° C., 5% CO2, 100% humidity. Treatmentwith 5, 10 and 20 μmon Tubastatin A (Chemie Tek), 1-2 μmon Tubacin(Chemie Tek), or 2, 4, and 8 μmon Compound A-1 (Acetylon PharmaceuticalInc.) suspended in NRC media was started 24 hours later. The drugvehicle, DMSO, was suspended in NRC Media as a control. Cellproliferation was assessed with the CellTiter 96 AQueous One Solution(MTS; Promega) and/or counting cells using the Cellometer Auto T4 CellCounter (Nexcelom Bioscience).

3D-Culture

Freshly isolated bile ducts from PCK rats were embedded in a rat-tailtype I Collagen matrix (BD Biosciences) and grown in the presence orabsence of 10 μmon Tubastatin A and 2 μmon Tubacin. Images of thegrowing cysts were taken every day over a period of 5 days and cyst sizewas measured with the software “Image J” (National Institute of Health).The circumferential areas for each cystic structure was compared to day0 to calculate percentages of growth as previously described (see Muffet al. Development and characterization of a cholangiocyte cell linefrom the PCK rat, an animal model of Autosomal Recessive PolycysticKidney Disease, Lab Invest 2006, 86:940-950; Masyuk et al. Octreotideinhibits hepatic cystogenesis in a rodent model of polycystic liverdisease by reducing cholangiocyte adenosine 3′,5′-cyclic monophosphate,Gastroenterology (2007) 132:1104-1116 27; Leeet al. MicroRNA15amodulates expression of the cell-cycle regulator Cdc25A and affectshepatic cystogenesis in a rat model of polycystic kidney disease, J.Clin. Invest. (2008) 118:3714-3724 28. Gradilone et al. Activation ofTrpv4 reduces the hyperproliferative phenotype of cystic cholangiocytesfrom an animal model of ARPKD, Gastroenterology (2010) 139:304-314e302).

PCK cholangiocytes proliferate at a higher rate than control ratcholangiocytes (see Banales J M, Masyuk T V, Gradilone S A, Masyuk A I,Medina J F, LaRusso N F: The cAMP effectors Epac and protein kinase a(PKA) are involved in the hepatic cystogenesis of an animal model ofautosomal recessive polycystic kidney disease (ARPKD), Hepatology 2009,49:160-174). To test if up regulation of HDAC6 contributes tocholangiocyte hyperproliferation, the HDAC6 selective inhibitortubastatin-A was added to cultured cholangiocytes.

FIG. 2A shows that tubastatin-A decreased PCK cholangiocyteproliferation in a dose and time dependent fashion. By day 3,proliferation of treated PCK cholangiocytes was reduced by 2.5-, 3.5-,and 4.15-folds for 5 μM, 10 μM and 20 μM tubastatin A, respectively.Additionally, a different HDAC6 specific inhibitor, tubacin, alsoreduced PCK cholangiocyte proliferation by 2.94-folds (FIG. 2B). Bothdrugs also decreased proliferation of cultured human ADPKDcholangiocytes by 85% and 16%, respectively (FIG. 5A). To furtherconfirm the effect of HDAC6 inhibition on proliferation, HDAC6 inhibitortubastatin-A significantly reduces cystic cholangiocyte proliferation asmeasured using a Cellometer Auto T4 Cell (FIG. 5B).

Liver cysts isolated from the PCK rat and grown in a 3-D collagen matrixwere treated with HDAC6 inhibitors (FIG. 2C). Analysis of thecircumferential areas of the PCK cystic structures showed significantinhibition of cyst growth. After four days of drug administration,tubastatin-A and tubacin reduced cyst growth by 131% and 125%,respectively compared to untreated cysts (FIG. 2D).

Example 5: Inhibition of HDAC6 Decreases β-Catenin Protein Levels

For a better understanding of the mechanisms that are responsible forthe inhibition of PCK cholangiocyte proliferation and cyst growth in 3Dculture, cultured PCK cholangiocytes were treated with tubastatin-A andtubacin and the amount of acetylated α-tubulin and β-catenin wasdetermined by Western blotting.

PCK cholangiocytes treated with tubastatin-A and tubacin had increasedlevels of acetylated α-tubulin (13- and 2.3-fold) compared to untreatedcells (FIG. 3A). In contrast, the levels of β-catenin were decreased by2.2-, and 5-fold after treatment with tubastatinA and tubacin,respectively (FIG. 3A).

Furthermore, immunofluorescence analysis of b-catenin subcellularlocalization also demonstrated that HDAC6 treatment decreased b-cateninexpression both in nuclei and cytoplasm (FIG. 6). Consistent withβ-catenin reduction, HDAC6 inhibition also caused a decrease in theβ-catenin gene target products, cyclin D1 and c-myc, while the levels ofacetylated histone H3 remained unaffected (FIG. 3A).

HDAC6 inhibition also increased β-catenin acetylation andphosphorylation (FIG. 3B), consistent with b-catenin destabilization andtargeting to degradation. β-catenin protein decreases over time whencells were treated with the HDAC6 inhibitor, while the levels ofβ-catenin messenger RNA remain stable, suggesting that HDAC6 inhibitioninduces the degradation of β-catenin protein (FIG. 3C).

Example 6: The Specific HADC6 Inhibitor, Compound A-1, Decreases CystFormation In Vivo Rat Pld Model.

The PCK rat model was used because the genetic mutation in this animalis orthologous to that found in human ARPKD. These animals express manyof the characteristics of human ADPKD (Lager D J, et al., KidneyInternat 59: 126-136, 2001; Harris, Curr Opin Nephrol Hypertens11:309-314, 2002). The animals carrying this mutation present with bothkidney and liver fibrocystic disease and these animals live long enoughto facilitate long-term treatment protocols (Gattone V. H., et al., NatMed (2003) 9: 1323-1326; Tones V. E., et al., Nat Med (2004) 10:363-364;Masyuk T. V., et al., Gastroenterology (2007) 132:1104-1116, 2007.Female animals show more severe liver disease than male animals.

Normal and PCK cholangiocytes were treated with the HDAC6 specificinhibitor, Compound A-1. PCK cells proliferation was inhibited by theCompound A-1 in a dose dependent manner, and had no significant effecton the proliferation of normal cells (FIG. 4A).

The efficacy of the Compound A-1 was then tested in a PCK rat, an animalmodel of polycystic liver disease. Both liver and kidney cyst area weresignificantly reduced by the treatment with the Compound A-1 [by 28.4%and 39.0%, respectively (see FIGS. 4B and 4C). Importantly, liverfibrosis was also reduced by 30.5% (p<0.001).

Example 7: The Specific HDAC6 Inhibitor of Compounds B-1 and C-2Decrease Cyst Formation In Vivo

Male PKC-1 rats (4 weeks old, n=12) were treated by oral gavage oncedaily with the indicated dose of compounds for 12 weeks. The animalswere sacrificed and kidneys were removed and fixed in formalin. Thevolume of cysts in the kidneys was measured by histological analysis(see FIG. 7). As a comparison, 6 rats were sacrificed at 4 weeks tomeasure the starting volume of renal cysts (control).

The results show that the HDAC6-specific inhibitor compounds B-1 and C-2decrease cyst formation in vivo.

1. A method for treating a polycystic disease comprising: administeringto a subject with a polycystic disease a therapeutically effectiveamount of a histone deacetylase 6 (HDAC6) specific inhibitor compound ofFormula I:

or a pharmaceutically acceptable salt thereof, wherein, ring B is arylor heteroaryl; R1 is an aryl or heteroaryl, each of which may beoptionally substituted by OH, halo, or C₁₋₆-alkyl; and R is H orC₁₋₆-alkyl or a therapeutically effective amount of a histonedeacetylase 6 (HDAC6) specific inhibitor compound of Formula II:

or a pharmaceutically acceptable salt thereof, wherein, X is C or O;R_(y) is independently, at each occurrence, selected from the groupconsisting of C₁₋₆-alkyl, C₁₋₆-alkoxy, halo, —C₁₋₆ haloalkyl, —O₁₋₆dihaloalkyl, —C₁₋₆ trihaloalkyl, —OH, —N(R¹)₂, —C(O)R¹, —CO₂R¹, and—C(O)N(R¹)₂; or: two R_(y) groups on the same or adjacent carbon atomsare taken together to form a C₃₋₈-cycloalkyl or O₃₋₇-heterocycloalkylring, each of which may be fused or isolated; R_(z) is independently, ateach occurrence, selected from the group consisting of C₁₋₆-alkyl,C₁₋₆-alkoxy, halo, —C₁₋₆ haloalkyl, —O₁₋₆ dihaloalkyl, —C₁₋₆trihaloalkyl, —OH, —N(R²)₂, —C(O)R², —CO₂R², —C(O)N(R²)₂, each R¹ isindependently, at each occurrence, selected from the group consisting ofH, C₁₋₆-alkyl, C₃₋₈-cycloalkyl, C₃₋₇-heterocycloalkyl, aryl, heteroaryl,C₁₋₆-alkyl-cycloalkyl, C₁₋₆-alkyl-heterocycloalkyl, C₁₋₆-alkyl-aryl, andC₁₋₆-alkyl-heteroaryl; each R² is independently, at each occurrence,selected from the group consisting of H or C₁₋₆-alkyl; m is 0, 1, 2, or3; and n is 0, 1, 2, or
 3. 2. The method for treating polycystic diseaseaccording to claim 1, wherein the amount of the histone deacetylase 6(HDAC6) specific inhibitor compound is effective at reducing cyst growthin the subject.
 3. The method for treating polycystic disease accordingto claim 1, wherein the polycystic disease is polycystic liver disease.4. The method for treating polycystic disease according to claim 2,wherein the cysts are located in the liver.
 5. The method for treatingpolycystic disease according to claim 1, wherein the polycystic diseaseis renal cystic disease.
 6. The method for treating polycystic diseaseaccording to claim 5, wherein the renal cystic disease is polycystickidney disease.
 7. The method for treating polycystic disease accordingto claim 2, wherein the cysts are located in the kidney.
 8. The methodfor treating a polycystic disease according to claim 5, wherein therenal cystic disease is an autosomal dominant polycystic kidney disease(ADPKD).
 9. The method for treating a polycystic disease according toclaim 5, wherein the renal cystic disease is autosomal recessivepolycystic kidney disease (ARPKD).
 10. The method for treating apolycystic disease according to claim 1, wherein the amount of thehistone deacetylase 6 (HDAC6) specific inhibitor compound is effectiveat preventing the formation of cysts.
 11. The method for treating apolycystic disease according to claim 1, wherein the amount of thehistone deacetylase 6 (HDAC6) specific inhibitor compound is effectiveat inhibiting cholangiocyte proliferation.
 12. The method for treating apolycystic disease according to claim 1, wherein the amount of thehistone deacetylase 6 (HDAC6) specific inhibitor compound is effectiveat increasing the amount of bile duct acetylated tubulin.
 13. The methodfor treating a polycystic disease according to claim 1, wherein theamount of the histone deacetylase 6 (HDAC6) specific inhibitor compoundis effective at reducing bile duct β-catenin synthesis.
 14. The methodfor treating a polycystic disease according to claim 1, wherein theamount of the histone deacetylase 6 (HDAC6) specific inhibitor compoundis effective at increasing bile duct β-catenin phosphorylation and/oracetylation.
 15. The method for treating a polycystic disease accordingto claim 1, wherein the histone deacetylase 6 (HDAC6) specific inhibitorcompound of Formula I is:

or a pharmaceutically acceptable salt thereof.
 16. The method fortreating a polycystic disease according to claim 15, wherein the amountof the histone deacetylase 6 (HDAC6) specific inhibitor compound iseffective at reducing cyst growth in the subject.
 17. The method fortreating a polycystic disease according to claim 15, wherein the amountof the histone deacetylase 6 (HDAC6) specific inhibitor compound iseffective at inhibiting cholangiocyte proliferation.
 18. The method fortreating a polycystic disease according to claim 1, wherein the histonedeacetylase 6 (HDAC6) specific inhibitor compound of Formula I is:

or a pharmaceutically acceptable salt thereof.
 19. The method fortreating a polycystic liver disease according to claim 18, wherein theamount of the histone deacetylase 6 (HDAC6) specific inhibitor compoundis effective at reducing cyst growth in the subject.
 20. The method fortreating a polycystic disease according to claim 18, wherein the amountof the histone deacetylase 6 (HDAC6) specific inhibitor compound iseffective at inhibiting cholangiocyte proliferation.
 21. (canceled) 22.(canceled)
 23. (canceled)